N-oxyde of n-phenyl-2-pyrimidine-amine derivatives

ABSTRACT

The invention relates to N-phenyl-2-pyrimidine-amine derivatives in which at least one nitrogen atom carries an oxygen atom to form the corresponding N-oxides, to processes for the preparation thereof, to pharmaceutical compositions comprising those compounds, and to the use thereof in the preparation of pharmaceutical compositions for the therapeutic treatment of warm-blooded animals, including humans.

The invention relates to N-phenyl-2-pyrimidine-amine derivatives inwhich at least one nitrogen atom carries an oxygen atom to form thecorresponding N-oxides, to processes for the preparation thereof, topharmaceutical compositions comprising those compounds, and to the usethereof in the preparation of pharmaceutical compositions for thetherapeutic treatment of warm-blooded animals, including humans.

The invention relates particularly to compounds of formula I

wherein

-   R₁ is hydrogen or hydroxy,-   R₂ is hydrogen, lower alkyl or hydroxy-lower alkyl,-   A is —NR₅R₆, —CR₅R₆ or —OR₅R₆,-   R₅R₆ together represent alkylene with four, five or six carbon    atoms, oxa-lower alkylene with one oxygen and three or four carbon    atoms, or aza-lower alkylene with one or two nitrogen and two, three    or four carbon atoms wherein the nitrogen atom is unsubstituted or    substituted by lower alkyl, hydroxy-lower alkyl, or acetyl, and    wherein lower alkylene in each case may be partially or totally    unsaturated and/or the carbon atoms of lower alkylene may be    substituted by lower alkyl, hydroxyl, lower alkoxy or oxo when lower    alkylene is not totally unsaturated, and    wherein at least one nitrogen atom carries an oxygen atom to form    the corresponding N-oxide or when no nitrogen atom carries an oxygen    atom, A is substituted by oxo on a ring carbon,    or a pharmaceutically acceptable salt of such a compound.

Preferably A is substituted by oxo on a ring carbon.

Preferably A is pyrrolidino, piperidyl, piperidino, piperazinyl,pyridyl, pyrrolidino, pyrrolidinyl, morpholino, lower alkylpiperazino,N-methylpiperazino, 4-methyl-3-oxo-1-piperazinyl, 3-oxo-1-piperazinyl,1H-imidazolyl, 1H-2-methylimidazolyl, 1H-4-methylimidazolyl or1H-2,4-dimethylimidazolyl, cyclohexyl or phenyl, optionally substitutedby oxo on a ring carbon;

Most preferably “A” represents a piperazino group of the followingformula A′

wherein

-   R₃ represents, hydrogen, lower alkyl or acetyl.

Preference is given to compounds of formula 1, wherein

-   R₁ is hydrogen,-   R₂ is hydrogen, methyl or hydroxymethyl,-   A is A′, optionally substituted by oxo on a ring carbon,-   R₃ is methyl or hydrogen,    or salts of such compounds.

When “A” is substituted by oxo on a ring carbon, “A” Is preferablyselected from lower alkyl-oxo-piperazino such as4-methyl-3-oxo-1-piperazinyl or oxo-piperazino such as3-oxo-1-piperazinyl, oxo-pyrrolidin, oxo-piperidino, oxo-piperidyl,oxo-morpholino, oxo-cyclohexyl, succinimido or glutarimido.

The nitrogen atoms, which carry an oxygen atom to form the correspondingN-oxides are preferably the ring nitrogen atoms located on pyrimidine,pyrindinyl, “A” or piperazino group of the formula A′.

By defining “R₅R₆ together”, the applicant does not include in thenumbering the nitrogen, oxygen or carbon group mentioned in NR₅R₆, CR₅R₆or OR₅R₆.

The prefix “lower” denotes a radical having up to and including amaximum of 7, especially up to and including a maximum of 4 carbonatoms, the radicals in question being either linear or branched withsingle or multiple branching.

Lower alkyl is preferably alkyl with from and including 1 up to andincluding 7, preferably from and including 1 to and including 4, and islinear or branched; preferably, lower alkyl is butyl, such as n-butyl,sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl,ethyl or methyl. Preferably lower alkyl is methyl, propyl or tert-butyl.

Hydroxy-lower alkyl, preferably hydroxymethyl, 2-hydroxyethyl or2-hydroxy-2-propyl.

Lower alkoxy is especially methoxy, ethoxy, isopropyloxy, ortert-butyloxy.

In a preferred aspect, the invention relates to compounds of formula II

wherein

-   R₁ is hydrogen or hydroxy,-   R₂ is lower alkyl or hydroxy-lower alkyl,-   R₃ is hydrogen, methyl or acetyl, and    the stars indicate the nitrogen atoms which optionally carry an    oxygen atom to form the corresponding N-oxides,    with the proviso that at least one of the three nitrogen atoms    marked by a star carries an oxygen atom if R₁ is hydrogen, R₂ is    methyl and R₃ is hydrogen or methyl, or salts of such compounds.

Optionally, the nitrogen atoms of the 2-pyrimidine can also carry one ortwo oxygen atoms to form the corresponding N-oxides.

Preferably, a compound of formula II carries at least one oxygen atom toform the corresponding N-oxide.

Optionally, the piperazinyl is substituted by oxo to from a loweralkyl-oxo-piperazino such as 4-methyl-3-oxo-1-piperazinyl or anoxo-piperazino such as 3-oxo-1-piperazinyl.

The term “lower” within the scope of compounds of formula II denotesradicals having up to and including 7, preferably up to and including 4carbon atoms.

When R₁ is hydroxy, the 3-pyridinyl moiety is substituted by hydroxy ata ring carbon atom at position 2, 4, 5 or 6.

Lower alkyl R₂ is preferably methyl.

Hydroxy-lower alkyl R₂ is preferably hydroxymethyl.

Salts are especially the pharmaceutically acceptable salts of compoundsof formula I or II.

Such salts are formed, for example, as acid addition salts, preferablywith organic or inorganic acids, from compounds of formula I or II witha basic nitrogen atom, especially the pharmaceutically acceptable salts.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. Only the pharmaceutically acceptable salts or freecompounds (if the occasion arises, in the form of pharmaceuticalcompositions) attain therapeutic use, and these are therefore preferred.

In view of the close relationship between the novel compounds in freeform and in the form of their salts, including those salts that can beused as intermediates, for example in the purification or identificationof the novel compounds, hereinbefore and hereinafter any reference tothe free compounds is to be understood as referring also to thecorresponding salts, as appropriate and expedient.

A compound of formula I or II possesses valuable pharmacologicalproperties and may, for example, be used as an anti-tumour agent, as anagent to treat atherosclerosis, as an agent to treat restenosis, as ananti-leukemic agent for the prevention of transplantation-induceddisorders, such as obliterative bronchiolitis, and/or for preventing theinvasion of warm-blooded animal cells by certain bacteria, such asPorphyromonas gingivalis.

The phosphorylation of proteins has long been known as an essential stepin the differentiation and division of cells. Phosphorylation iscatalysed by protein kinases subdivided into serine/threonine andtyrosine kinases. The tyrosine kinases include PDGF (Platelet-derivedGrowth Factor) receptor tyrosine kinase.

PDGF is a very commonly occurring growth factor, which plays animportant role both in normal growth and also in pathological cellproliferation, such as is seen in carcinogenesis and in diseases of thesmooth-muscle cells of blood vessels, for example in atherosclerosis andthrombosis.

The inhibition of PDGF-stimulated receptor tyrosine kinase activity invitro is measured in PDGF receptor immune complexes of A431 cells, asdescribed by E. Andrejauskas-Buchdunger and U. Regenass in CancerResearch 52, 5353-5358 (1992). A compound of formula I or II inhibitsPDGF-dependent acellular receptor phosphorylation. The inhibition ofPDGF receptor tyrosine kinase is measured in a microtitre ELISA assay(cf Trinks et al., J. Med. Chem. 37, 1015-27 (1994).

The inhibition of PDGF receptor tyrosine kinase makes a compound offormula I or II also suitable for the treatment of tumour diseases, suchas gliomas, sarcomas, prostate tumours, and tumours of the colon,breast, and ovary.

A compound of formula I or II also inhibits cellular processes involvingthe so-called stem-cell factor (SCF, also known as the c-Kit ligand orsteel factor), such as SCF receptor (Kit) autophosphorylation and theSCF-stimulated activation of MAPK kinase (mitogen-activated proteinkinase).

In particular, a compound of formula I or II inhibits the tyrosinekinase activity of c-Kit. This can be shown in a tyrosine kinaseinhibition assay using the cytoplasmatic kinase domain of c-Kit. Theassay is performed as follows: The baculovirus donor vector pFbacG01(GIBCO) is used to generate a recombinant baculovirus that expresses theamino acid region amino acids 544-976 of the cytoplasmic kinase domainsof human c-Kit. The coding sequences for the cytoplasmic domain of c-Kitis amplified by PCR from a human uterus c-DNA library (Clontech). Theamplified DNA fragment and the pFbacG01 vector are made compatible forligation by digestion with BamHI and EcoRI. Ligation of these DNAfragments results in the baculovirus donor plasmid c-Kit. The productionof the viruses, the expression of proteins in Sf9 cells and thepurification of the GST-fused proteins are performed as follows:Production of virus: Transfer vector (pFbacG01-c-Kit) containing thec-Kit kinase domain is transfected into the DH10Bac cell line (GIBCO)and the transfected cells are plated on selective agar plates. Colonieswithout insertion of the fusion sequence into the viral genome (carriedby the bacteria) are blue. Single white colonies are picked and viralDNA (bacmid) is isolated from the bacteria by standard plasmidpurification procedures. Sf9 cells or Sf21 cells (American Type CultureCollection) are then transfected in 25 cm² flasks with the viral DNAusing Cellfectin reagent.

Determination of small scale protein expression in Sf9 cells: Viruscontaining media is collected from the transfected cell culture and usedfor infection to increase its titre. Virus containing media obtainedafter two rounds of infection is used for large-scale proteinexpression. For large-scale protein expression 100 cm² round tissueculture plates are seeded with 5×10⁷ cells/plate and infected with 1 mLof virus-containing media (approx. 5 MOIs). After 3 days the cells arescraped off the plate and centrifuged at 500 rpm for 5 min. Cell pelletsfrom 10-20, 100 cm² plates, are resuspended in 50 mL of ice-cold lysisbuffer (25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1% NP-40, 1 mM DTT, 1 mMPMSF). The cells are stirred on ice for 15 min and then centrifuged at5000 rpms for 20 min.

Purification of GST-tagged protein: The centrifuged cell lysate isloaded onto a 2 mL glutathione-sepharose column (Pharmacia) and washedthree times with 10 mL of 25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM DTT,200 mM NaCl. The GST-tagged protein is eluted by 10 applications (1 mLeach) of 25 mM Tris-HCl, pH 7.5, 10 mM reduced-glutathione, 100 mM NaCl,1 mM DTT, 10% Glycerol and stored at −70° C.

Kinase assay: Tyrosine protein kinase assays with purified GST-c-Kit arecarried out in a final volume of 30 μL containing 200-1800 ng of enzymeprotein (depending on the specific activity), 20 mM Tris-HCl, pH 7.6, 3mM MnCl₂, 3 mM MgCl₂, 1 mM DTT, 10 μM Na₃VO₄, 5 μg/mL poly(Glu,Tyr) 4:1,1% DMSO, 1.0 μM ATP and 0.1 μCi [γ³³ P] ATP. The activity is assayed inthe presence or absence of inhibitors, by measuring the incorporation of³³P from [γ³³P] ATP into the poly(Glu,Tyr) 4:1 substrate. The assay (30μL) is carried out in 96-well plates at ambient temperature for 20 minunder conditions described below and terminated by the addition of 20 μLof 125 mM EDTA. Subsequently, 40 μL of the reaction mixture istransferred onto Immobilon-PVDF membrane (Millipore, Bedford, Mass.,USA) previously soaked for 5 min with methanol, rinsed with water, thensoaked for 5 min with 0.5% H₃PO₄ and mounted on vacuum manifold withdisconnected vacuum source. After spotting all samples, vacuum isconnected and each well rinsed with 200 μL 0.5% H₃PO₄. Membranes areremoved and washed 4× on a shaker with 1.0% H₃PO₄ and once with ethanol.

Membranes are counted after drying at ambient temperature, mounting inPackard TopCount 96-well frame, and addition of 10 μwell of Microscint™(Packard). IC₅₀ values are calculated by linear regression analysis ofthe percentage inhibition of each compound in duplicate, at fourconcentrations (usually 0.01, 0.1, 1 and 10 μM). One unit of proteinkinase activity is defined as 1 nmole of ³³P ATP transferred from [γ³³P]ATP to the substrate protein per minute per mg of protein at 37° C.

A compound of formula I or II inhibits also the autophosphorylation ofSCF receptor (and c-Kit, a proto-oncogen). Inhibition of theautophosphorylation of the SCF receptor can be measured using e.g. MO7ecells, a human promegakaryocytic leukaemia cell line which depends onSCF for proliferation. They are obtained from Grover Bagby, OregonHealth Sciences University, USA. The cells are cultivated in RPMI 1649medium supplemented with 10 FBS and 2.5 ng/ml GC-CMF. GM-SCF and SCF arecommercially available. Serum-deprived MO7e cells are prepared andincubated for 90 min at 37° C. with the test substance before beingstimulated with recombinant SCF for 10 min at 37° C. Identicalquantities of cell lysates are analysed by Western blot usingantiphosphotyrosine antibodies (Buchdunger et al., Proc. Natl. Acad. Sci(USA) 92, 2558-62 (1995)). The immunodecorated proteins are detected bymeans of the ECL Western blotting system from Amersham (Amersham, UK).

On the basis of the described properties, a compound of formula I or IImay be used not only as a tumour-inhibiting substance, for example insmall cell lung cancer, but also as an agent to treat non-malignantproliferative disorders, such as atherosclerosis, thrombosis, psoriasis,scieroderma, and fibrosis, as well as for the protection of stem cells,for example to combat the haemotoxic effect of chemotherapeutic agents,such as 5-fluoruracil, and in asthma. It may especially be used for thetreatment of diseases which respond to an inhibition of the PDGFreceptor kinase.

In addition, a compound of formula I or II prevents the development ofmultidrug resistance in cancer therapy with other chemotherapeuticagents or abolishes a pre-existing resistance to other chemotherapeuticagents. Also regardless of the effect described hereinbefore, a compoundof formula I or II may be used to advantage in combination with otherantitumour agents, such as especially other c-Kit inhibitors andinhibitors of Vascular Endothelial Growth Factor (VEGF) receptor orc-Src activity.

Also Abl kinase, especially v-Abl kinase, is inhibited by a compound offormula I or II. The inhibition of v-Abl tyrosine kinase is determinedby the methods of N. Lydon et al., Oncogene Research 5, 161-173 (1990)and J. F. Geissler et al., Cancer Research 52, 4492-8 (1992). In thosemethods [Val⁵]-angiotensin II and [γ-³²P]-ATP are used as substrates.

By analogy, a compound of formula I or II also inhibits Bcr-AbI kinase(see Nature Medicine 2, 561-566 (1996)) and is thus suitable for thetreatment of Bcr-Abl-positive cancer and tumour diseases, such asleukaemias (especially chronic myeloid leukaemia and acute lymphoblasticleukaemia, where especially apoptotic mechanisms of action are found),and also shows effects on the subgroup of leukaemic stem cells as wellas potential for the purification of these cells in vitro after removalof said cells (for example, bone marrow removal) and reimplantation ofthe cells once they have been cleared of cancer cells (for example,reimplantation of purified bone marrow cells).

Test for activity against c-Abl protein tyrosine kinase. The test isconducted as a filter binding assay as follows: The His-tagged kinasedomain of c-Abl is cloned and expressed in the baculovirus/Sf9 system asdescribed by Bhat et al., J. Biol. Chem. 272, 16170-5 (1997). A proteinof 37 kD (c-Abl kinase) is purified by a two-step procedure over acobalt metal chelate column followed by an anion exchange column with ayield of 1-2 mg/L of Sf9 cells.

The purity of the c-Abl kinase is >90% as judged by SDS-PAGE afterCoomassie blue staining. The assay contains: c-Abl kinase (50 ng), 20 mMTris-HCl, pH 7.5, 10 mM MgCl₂, 10 μM Na₃VO₄, 1 mM DTT and 0.06 μCi/assay[γ³³ P]-ATP (5 μM ATP) using 30 μg/mL poly-Ala,Glu,Lys,Tyr-6:2:5:1(Poly-AEKY, Sigma P1152) in the presence of 1% DMSO, total volume of 30μL. Reactions are terminated by adding 10 μL of 250 mM EDTA, and 30 μLof the reaction mixture is transferred onto Immobilon-PVDF membrane(Millipore, Bedford, Mass., USA) previously soaked for 5 min withmethanol, rinsed with water, then soaked for 5 min with 0.5% H₃PO₄ andmounted on vacuum manifold with disconnected vacuum source. Afterspotting all samples, vacuum is connected and each well rinsed with 200μL 0.5% H₃PO₄. Membranes are removed and washed on a shaker with 0.5%H₃PO₄ (4 times) and once with ethanol. Membranes are counted afterdrying at ambient temperature, mounting In Packard TopCount 96-wellframe, and addition of 10 μL/well of Microscint™ (Packard).

Test for activity against Bcr-Abl. The murine myeloid progenitor cellline 32Dcl3 transfected with the p210 Bcr-Abl expression vectorpGDp210Bcr/Abl (32D-bcr/abl) was obtained from J. Griffin (Dana FaberCancer Institute, Boston, Mass., USA). The cells express the fusionBcr-Abl protein with a constitutively active Abl kinase and proliferategrowth factor independent. The cells are expanded in RPMI 1640 (AMIMED),10% fetal calf serum, 2 mM glutamine (Gibco) (“complete medium”), and aworking stock is prepared by freezing aliquots of 2×10⁶ cells per vialin freezing medium (95% FCS, 5% DMSO (SIGMA)). After thawing, the cellsare used during maximally 10-12 passages for the experiments.

For cellular assays, compounds are dissolved in DMSO and diluted withcomplete medium to yield a starting concentration of 10 μM followed bypreparation of serial 3-fold dilutions in complete medium. 200'00032D-Bcr/Abl cells in 50 μL complete medium are seeded per well in 96well round bottom tissue culture plates. 50 μL per well of serial 3-folddilutions of the test compound are added to the cells in triplicates.Untreated cells are used as control. The compound is incubated togetherwith the cells for 90 min at 37° C., 5% CO₂, followed by centrifugationof the tissue culture plates at 1300 rpm (Beckman GPR centrifuge) andremoval of the supernatants by careful aspiration taking care not toremove any of the pelleted cells. The cell pellets are lysed by additionof 150 μL lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM sodium chloride,5 mM EDTA, 1 mM EGTA, 1% NP-40, 2 mM sodium ortho-vanadate, 1 mM PMSF,50 μg/mL aprotinin and 80 μg/mL leupeptin) and either used immediatelyfor the ELISA or stored frozen in the plates at −20° C. until usage.

Black ELISA plates (Packard HTRF-96 black plates) are precoated overnight at 40C with 50 ng/well of the rabbit polyclonal anti-abl-SH3domain Ab 06-466 from Upstate in 50 μL PBS. After washing 3 times with200 μL/well PBS containing 0.05% Tween20 (PBST) and 0.5% TopBlock(Juro), residual protein binding sites are blocked with 200 μL/wellPBST, 3% TopBlock for 4 h at room temperature followed by incubationwith 50 μL lysates of untreated or compound-treated cells (20 μg totalprotein per well) for 3-4 h at 4° C. After 3 washings, 50 μL/wellanti-phosphotyrosine Ab PY20(AP) labeled with alkaline phosphatase(Zymed) diluted to 0.2 μg/mL in blocking buffer is added and incubatedover night (4° C.). For all incubation steps the plates are covered withplate sealers (Costar). Finally, the plates are washed another threetimes with washing buffer and once with deionized water before additionof 90 pUwell of the AP-substrate CDPStar RTU with Emerald II. Theplates, now sealed with Packard TopSeal™-A plate sealers, are incubatedfor 45 min at room temperature in the dark and luminescence isquantified by measuring counts per second (CPS) with a Packard Top CountMicroplate Scintillation Counter (Top Count). The difference between theELISA-readout (CPS) obtained for with the lysates of the untreated32D-Bcr/Abl cells and the readout for the assay-background (allcomponents, but without cell lysate) is calculated and taken as 100%reflecting the constitutively phosphorylated Bcr-Abl protein present inthese cells. The activity of the compound on the Bcr-Abl kinase activityis expressed as percent reduction of the Bcr-Abl phosphorylation. Thevalues for the IC₅₀ and IC₉₀ are determined from the dose responsecurves by graphical extrapolation.

Test for activity against mutant Bcr-Abl: The activity of compounds onthe M351T mutant Bcr-Abl kinase activity is assessed as described above,except that 32Dcl3 cells transfected with mutant Bcr-Abl in place ofp210 Bcr-Abl are utilised.

c-Raf-1 protein kinase assay: Recombinant c-Raf-1 protein is obtained bytriple infection of Sf21 cells with GST-c-Raf-1 recombinant baculovirustogether with v-Src and v-Ras recombinant baculoviruses that arerequired for active c-Raf-1 kinase production (Williams et al., PNAS1992; 89:2922-6). Active Ras (v-Ras) is required to recruit c-Raf-1 tothe cell membrane and v-Src to phosphorylate c-Raf-1 to fully activateit. Cells are seeded at 2.5×10⁷ cells per 150 mm dish and allowed toattach to a 150 mm dish for 1 hr at RT. Media (SF90011 containing 10%FBS) is aspirated and recombinant baculovirus GST-c-Raf-1, v-Ras andv-Src are added at MOI of 3.0, 2.5 and 2.5, respectively, in a totalvolume of 4-5 mL. Cells are incubated for 1 hr at RT and then 15 mL ofmedium is added. Infected cells are incubated for 48-72 hr at 270C.Infected Sf21 cells are scraped and collected into a 50 mL tube andcentrifuged for 10 min at 4° C. at 1100 g in a Sorvall centrifuge. Thecell pellet is washed once with ice cold PBS and lysed with 0.6 mL lysisbuffer per 2.5×10⁷ cells. Complete lysis of cells is achieved after 10min on ice with occasional pipetting. The cell lysates are centrifugedfor 10 min at 40C at 14,500 g in a Sorvall centrifuge with SS-34 rotorand the supernatant is transferred to a fresh tube and stored at −80° C.c-Raf-1 is purified from cell lysates using 100 μL of packedglutathione-sepharose 4B beads equilibrated in ice cold PBS per 2.5×10⁷cells. GST-c-Raf-1 is allowed to bind to the beads at 40C for 1 hr withrocking. Bound GST-c-Raf-1 with beads is transferred to a column. Thecolumn is washed once with lysis buffer and twice with ice cold Trisbuffered saline. Ice cold elution buffer is added and column flow isstopped to allow the free glutathione to disrupt the interaction ofGST-c-Raf-1 with glutathione sepharose beads. Fractions (1 mL) arecollected into pre-chilled tubes. Each tube contains 10% glycerol (finalconcentration) to maintain kinase activity during freeze thaw cycles.Purified fractions of GST-c-Raf-1 kinase protein are stored at −80° C.

IκB is used as substrate for the c-Raf-1 kinase. IκB is expressed inbacteria as a His-tagged protein BL21. LysS bacteria containing the IκBplasmid are grown to an OD600 of 0.6 in LB medium, then induced toexpress the IκB with IPTG (final concentration of 1 mM) for 3 hrs at370C and then bacteria are lysed by sonication (microtip limit settingfor 3 times at 1 min each in sonication buffer [50 mM Tris pH 8.0, 1 mMDTT, 1 mM EDTA] and centrifuged at 10,000 g for 15 min. The supernatantis mixed with ammonium sulfate to give a final concentration of 30%.This mixture is rocked for 15 min at 4 C then spun at 10,000 g for 15min. The pellet is resuspended in binding buffer (Novagen) containing 10mM BSA. This solution is applied to Ni-agarose (Novagen) and washedaccording to the Novagen manual. IκB is eluted from the column usingelution buffer (0.4 M imidazole, 0.2 M NaCl, 8 mM Tris pH 7.9).Fractions containing protein are dialysed in 50 mM Tris pH 8, 1 mM DTT.

The activity of c-Raf-1 protein kinase is assayed in the presence orabsence of inhibitors, by measuring the incorporation of ³³P from [y33p]ATP into IκB. The assay is carried out in 96-well plates at ambienttemperature for 60 min. It contains (total volume of 30 μL): c-Raf-1kinase (400 ng), 25 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 5 mM MnCl₂, 10 μMNa₃VO₄, 1 mM DTT and 0.3 μCi/assay [y33 P]-ATP (10 μM ATP) using 600 ngIκB in the presence of 1% DMSO. Reactions are terminated by adding 10 μLof 250 mM EDTA and 30 μL of the reaction mixture is transferred ontolmmobilon-PVDF membrane (Millipore, Bedford, Mass., USA) previouslysoaked for 5 min with methanol, rinsed with water, then soaked for 5 minwith 0.5% H₃PO₄ and mounted on vacuum manifold with disconnected vacuumsource. After spotting all samples, vacuum is connected and each wellrinsed with 200 μL 0.5% H₃PO₄. Membranes are removed and washed 4× on ashaker with 0.5% H₃PO₄, once with ethanol. Membranes are counted afterdrying at ambient temperature, mounting in Packard TopCount 96-wellframe, and addition of 10 μL/well of Microscint™ (Packard).

It has also been surprisingly discovered that our compounds of formula Ior II have an unexpected potential to serve as hypoxia-selectiveproducts due to bio-reduction (deoxygenation) in the cell especially inthe tumours and in the brain. Hypoxia-activated pro-drugs are especiallyuseful in cancer therapy since severe hypoxia occurs in solid tumourtissue or in the brain. Hypoxic cells can be exploited for therapy bynon-toxic, hypoxia-activated pro-drugs. Thus because of the netreduction of the N-oxide moiety, there is an higher uptake of compoundsof formula I or II in the tumours or in the brain and an accumulation ofthe reduced form of the compounds of formula I or II in the tumours orbrain.

Another advantage of the compounds of formula I or II, is superioreffects to carrier-mediated efflux over compound A (by a saturablesystem, probably P-gp). Consequences of this less pronounced efflux is;

-   -   a greater absorption    -   higher drug levels in the brain and    -   higher drug levels in the tumour.        This effect on P-gp and transport mechanism may be demonstrated        as follows:

Caco-2 cell monolayers grown on polyethylene terephthalate (PET) filters(Falcon™) for 21-25 days are used for transport experiments. The flux ofcompounds across Caco-2 cell monolayers grown on PET filters as well asacross PET filters alone without Caco-2 cells (for system validation) inthe presence and absence of the potent efflux pump inhibitors CsA andVerapamil, respectively, are determined as follows: Prior to thetransport experiment, the culture medium in the acceptor compartment(0.2 ml for apical and 1.0 ml for basolateral sides) is replaced withacceptor solution (HBSS, when relevant containing the inhibitor ofinterest) preincubated at 37° C. To start the experiment, the medium inthe donor compartment (0.35 ml for apical and 1.15 ml for basolateralsides) is replaced with donor solution (compound in HBSS, when relevantcontaining inhibitor of interest) preincubated at 37° C. Aliquots of 150μl are removed from the donor and the acceptor side after about 1 and120 minutes. Transport experiments in both apical-to-basolateral andbasolateral-to-apical directions are performed in triplicate at 37° C.in an incubator without shaking.

Furthermore, the plasma protein binding of the compounds of formula I orII is superior with regard to free fraction and/or association withplasma proteins (e.g. albumin, α-1-acid glycoprotein (MG)), to thatobserved with compound A. A lower extent of association to MG results inless pronounced variability of free fraction of N-oxides and also has aneffect of free fraction of compound A compounds. At the clinicallyrelevant dose of 400 mg daily dose (concentrations of 900-2600 ng/mL ofcompound A), the free fraction of compound A ranges from 4 to 5%. Usingerythrocyte partitioning, compound A was mainly found associated withalbumin and alpha-1-acid glycoprotein (MG). The fraction associated withlipoproteins and gamma globulins was <5%. The reduced plasmaprotein-binding of compounds of formula I or II is shown by thefollowing example.

The free (or unbound) fraction of compounds of formula I or II isdetermined by the ultracentrifugation method which was used also forcompound A (see European patent application No. 1250140 or Internationalpatent application WO 01/47507 filed on 22/12/2000). Solutions of humanserum albumin (40 g/L) and α-1-acid glycoprotein (1 g/L) will beprepared in Soerensen buffer pH 7.4 containing 0.9% NaCl (w/v). 30 μL ofthe compounds of formula I or II stock solutions are directly spikedinto 3 mL of the protein solutions to get the intended finalconcentrations of 300-5000 ng/mL compounds of formula I or II (ethanolfinal concentration 0.5%, factor 1:200). After incubation for 30 min at37° C. under constant gentle agitation, the spiked protein samples(n=3/4) are centrifuged at 200'000 g for at least 5 hours h at 37° C.(centrifuge with a fixed angle rotor) using thick-walled polycarbonatecentrifuge tubes. The spin is stopped without braking. The concentrationof compounds of formula I or II are determined after incubation (beforecentrifugation) and after centrifugation in the supernatant.

The pharmacokinetics of the compounds of formula I or II areadvantageous over compound A with regard to C_(max) (highest observedconcentration in plasma in units mass/volume), half-live (refers to thetime after administration of the drug to observe a diminution of onehalf of the measured pharmacological response; In one aspect, thehalf-life is enhanced when the half-life is increased by at least 50%)or AUC (plasma concentrations over time, as defined by the Area Underthe Curve (AUC) in units of mass-time/volume) in plasma as a matter oftransport mechanism (e.g. P-gp). This advantageous pharmacokinetics isshown by giving to animals (e.g. rats) a single dose of compounds offormula I or II (one group of animals will be treated intravenously andone group of animals will be treated per orally). Blood is taken atselected time points (e.g. 0.083 min (iv: intra venous) and 0.25, 0.5,1, 2, 4, 6, 8, 10, 12, 24 and 48 h after iv or po (per orally dosing).

Plasma is prepared immediately by centrifugation of blood. Unchangedcompounds of formula I or II and compound A are measured in plasma usingHPLC/UV detection or LC-MS.

Furthermore, the reduced plasma protein binding of the compounds offormula 1 or II to plasma proteins can cause an increase in the apparentvolume of distribution due to higher fraction of unbound drug (fu).Advantageously, the distribution of the compounds of formula I or IIinto organs and tissues (including the brain) is different from that ofcompound A. This can be shown as follows.

-   -   Compounds of formula I or II and compound A in brain of mice or        rats upon dosing with non-radiolabelled N-oxide.

Animals (e.g. mice or rats) receive a single dose of compounds offormula I or II (one group of animals is treated intravenously and onegroup of animals is treated per orally). Animals are sacrificed atselected time points (e.g. 0.083 min (iv) and 1, 8, 24 and 48 h after ivor po dosing). Brain of treated animals are taken, homogenates of brainare prepared and samples are prepared (e.g. extraction of homogenatewith organic solvent such as methanol, acetonitrile or others) foranalysis (HPLC/UV or LC-MS) of compounds of formula I or II and compoundA. Concentrations of compounds of formula I or II and compound A inbrain and plasma is measured for determination of ratio brain/plasma.

-   -   Distribution of radioactive substance(s) in mice or rats upon        dosing with radiolabelled N-oxide.

For the tissue distribution study, e.g. 10 mg/kg po of radiolabelledN-oxide (e.g. [¹⁴C]-label; 100 μCi/kg b.w.) is administered to animals.The uptake/distribution of radioactive substance(s) throughout the bodyof the animal is investigated using quantitative whole-bodyautoradioluminography (QWABL). The animals arer sacrificed at selectedtime points and frozen in a mixture of dry-ice and hexane at approx.−75° C. Frozen animals are embedded in a pre-cooled 2% aqueous gel ofNa-CMC, at approx. −75° C.; 40 μm thick sections are obtained at ca.−20° C. in a CryoMacrocut cryomicrotome (Leica lnstr. GmbH, D-Nussloch).Dehydration of sections take place during 24-60 h at −23° C. in thecryomicrotome. Sections exposure to BAS III Imaging plates (Fuji PhotoFilm Co., Ltd., J-Tokyo) for 1 day at room temperature in a leadshielding box to minimize the increase of the background. The durationof exposure allows detection of ca. 2 dpm/mg, i.e. the radioactivityconcentration corresponding to ca. 0.2-0.4% of the total radioactivedose if the radioactivity was evenly distributed throughout the body.Scan is performed in a Fuji BAS 2000 TR phosphor imager, immediatelyafter the end of the exposure, under controlled light conditions, at a100 μm scanning step with a 1024 gradation. Image analysis is done asfollows: The resulting photostimulated light data files are corrected bysubtracting the background, processed electronically with the help of aMCID/M4 (3.0 Rev. 1.3) image analyzer (Imaging Research, St. Catherines,Ontario, Canada) and automatically converted into radioactivityconcentrations using a 1st degree polynomial calibration curve obtainedfrom a radioactive blood scale processed under similar conditions as thesamples. Detection (LD) and quantitation (QL) limits are determined byLD=mean of background (n=10)+3 SD; QL=3 LD.

The size of the measurement areas are the same as that of each bloodstandard of the blood scale used to set the calibration curve. Imagefiles processed using the Adobe Photoshop® software.

For distribution of total radioactive substance(s) Using QWABL only halfof the treated animal are used which makes possible to use organs ortissues for determination of unchanged N-oxide and/or compound A inselected samples using HPLC/UV or HPLC-radioactivity and/or LC-MS.

Compounds of formula I or II have also less affinity to CYP450s [1, 2],because of N-oxides which are more polar. These enzymes are metabolizingmost of the drugs on the market. Less affinity translates to smallerdrug/drug interaction potential. Especially the blocking of a basicnitrogen like in a piperazine/pyridine moiety reduce the affinity toCYP2D6 an enzyme which binds substrates especially by ionic interactionwith the aspartate residue which requires a basic moiety like thenitrogen in the piperazine ring system. A pool out of ten differenthuman liver microsomes are incubated with all cofactors necessary fortheir metabolic activity (NADPH) with defined marker substrates for therespective CYP450 isozyme specific activity. The potential inhibitor isadded with increasing concentrations and the metabolic reactions areevaluated by the corresponding analytical method (LC/MS, HPLC,Fluorescence). The conversion rate without inhibitor is set to 100% andthe inhibition rate is evaluated as the concentration of inhibitorneeded to suppress 50% of the conversion (IC50). The following markersubstrates are used: Substrates CYP Preferred Acceptable 1A2Ethoxyresorufin Caffeine (low turnover) Phenacetin Theophylline (lowturnover) Acetanilide (mostly applied in hepatocytes) Methoxyresorufin2A6 Coumarin 2C8 Paclitaxel (availability of standards?) 2C9 S-WarfarinTolbutamide (low Diclofenac turnover) 2C19 S-Mephenytoin (4-hydroxymetabolite) Omeprazole 2D6 Bufuralol Metoprolol DextromethorphanDebrisoquine Codeine (all with no problems, but less commonly used) 2E1Chlorzoxazone 4-Nitrophenol Lauric Acid 3A4 Midazolam NifedipineTestosterone (strongly Felodipine recommended to use at Cyclosporinleast two structurally Terfenadine unrelated substrates) ErythromycinSimvastatin

In addition, a compound of formula I or II shows useful effects in thetreatment of disorders arising as a result of transplantation, forexample, allogenic transplantation, especially tissue rejection, such asespecially obliterative bronchiolitis (OB), i.e. a chronic rejection ofallogenic lung transplants. In contrast to patients without OB, thosewith OB often show an elevated PDGF concentration in bronchoalveolarlavage fluids. If a compound of formula I or II is administered to ratswith tracheal allogenic transplants, for example in a dose of 50 mg/kgi.p., it can be shown after removal of 10 transplants per group after 10and 30 days for morphometric analysis of possible epithelial lesions andocclusion of the airways, and investigation for immunohistochemicalpathways of action that, although a compound of formula I or II has nosignificant effect on epithelial necrosis or infiltration byinflammatory cells, it does markedly reduce fibroproliferation andocclusion of the lumen compared with controls. Synergistic effects withother immunomodulatory or anti-inflammatory substances are possible, forexample when used in combination with cyclosporin A (CsA), rapamycin, orascomycin, or immunosuppressant analogues thereof, for examplecyclosporin G, FK-506 or comparable compounds; corticosteroids;cyclophosphamide; azathioprine; methotrexate; brequinar; leflunomide;mizoribine; mycophenolic acid; mycophenolate mofetil;15-deoxyspergualin; immunsuppressant antibodies, especially monoclonalantibodies for leucocyte receptors, for example MHC, CD2, CD3, CD4, CD7,CD25, CD28, B7, CD45, CD58 or their ligands; or other immunomodulatorycompounds, such as CTLA41g. If CsA (1 mg/kg s.c.), for example, iscombined with a compound of formula I or II (50 mg/kg), synergism may beobserved.

A compound of formula I or II is also effective in diseases associatedwith vascular smooth-muscle cell migration and proliferation (where PDGFand PDGF receptor often also play a role), such as restenosis andatherosclerosis. These effects and the consequences thereof for theproliferation or migration of vascular smooth-muscle cells in vitro andin vivo can be demonstrated by administration of a compound of formula Ior II and also by investigating its effect on the thickening of thevascular intima following mechanical injury in vivo.

A compound of formula I or II is used in 0.1 N HCl or DMSO at aconcentration of 10 mM for in vitro studies. The stock solution isfurther diluted with cell culture medium and used In concentrations of10 to 0.1 μM for the experiments. For in vivo administration, a compoundof formula I or II is dissolved for example in DMSO at a concentrationof 200 mg/ml and then diluted 1:20 with 1% Tween in 0.9% salinesolution. After sonication, a clear solution Is obtained. The stocksolutions are prepared fresh each day before administration. (Thecompound of formula I or II may also be dissolved simply in deionisedwater for oral administration or in 0.9% saline solution for parenteraladministration). Administration is carried out 24 hours before theoperation. A compound of formula I or II is administered to rats in onedose of 50 mg/kg i.p. per day for the entire observation period. Controlrats are given the same formulation but without the presence of acompound of formula I or II. Oral administration is also possible.

Primary cultures of smooth-muscle aorta cells are isolated from 9 to11-day-old DA (AG-B4, RT1a) rat aorta using a modification of the methoddescribed by Thyberg et al. (see Differentiation 25, 156-67 (1983)). Theaorta is opened by means of a longitudinal incision and the endotheliumcarefully removed. The adventitia and the tunica media are separated,and the tunica media is digested with 0.1% colagenase and DNAse inphosphate-buffered physiological saline for 30 min at 37° C. The cellsare centrifuged, suspended in culture medium, and then allowed to growon plastic vials. The primary cells are used for the experiments afterpassages 2 to 6. Subcultures are kept in DMEM (Dulbecco's ModifiedEagle's Medium), supplemented with 10% fetal calf serum, 2 mmol/mlglutamine, 100 mmol/ml streptomycin, and 100 IU/ml penicillin. Foridentification purposes, the cells are left to grow on glass slidecovers and stained immunohistochemically using an anti-α-actin antibodyobtained from smooth-muscle cells (see below).

The migration of smooth-muscle cells is quantified in vitro using aTranswell cell culture insert (Costar, Cambridge, Mass.) whose upper andlower compartments are separated by a polycarbonate membrane of 8 μmpore size. The cells (100 μl at a concentration of 1 million cells/ml)are exposed in the upper compartment. After 2 hours, 60 ng/ml PDGF-BB orPDGF-M (Upstate Biotechnology Inc., Lake Placid, N.Y.) is added to thelower compartment, supplemented with 0.5% fetal calf serum and 0.1%bovine serum albumin, and the test compound of formula I or II is addedin concentrations of 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 μM. Tomeasure fibronectin-dependent migration, the Transwell chambers arecovered with fibronectin at a concentration of 10 μg/ml for 24 h at 4°C. (human cellular fibronectin, Upstate Biotechnology Inc.). After 24hours' migration, the filters are removed, fixed in methanol, andstained with Mayer's haematoxylin and eosin. The migrated cells on thelower side of the filter membrane are determined by counting thespecified sectional fields on the filters with the aid of a lightmicroscope with a magnification of 400×. The inhibition of migration isquantified in terms of the percentage of cells versus with the control.To exclude the possibility of a toxic effect, the viability of the cellsis tested by incorporation of 3H-thymidine in DMEM, supplemented with10% fetal calf serum. An inhibition of migration induced by PDGF-M andespecially by PDGF-BB is observed with a compound of formula I or II.

Experimental animals: the aorta and carotid artery of male Wistar rats(purchased from the Laboratory Animal Centre of the University ofHelsinki, Finland) are denuded. The rats are anaesthetised with 240mg/kg chloral hydrate i.p. and Buprenorphine (Temgesic, Reckitt &Coleman, Hull, UK) is administered for perioperative and postoperativealleviation of pain. All animals are given human care in keeping withthe “Principles of Laboratory Animal Care” and the “Guide for the Careand Use of Laboratory Animals” of the NIH (NIH Publication 86-23,revised 1985). Rats weighing 200-300 g were used for the denudationprocedure. The left common carotid artery is denuded of endotheliumthrough the intraluminal passage of a 2F embolectomy catheter (BaxterHealthcare Corporation, Santa Ana, Calif., 27). To remove theendothelium, the catheter is passed through the lumen three times,inflated with 0.2 ml air. The external carotid is ligated after removalof the catheter and the wound closed. The histological changes areevaluated by reference to sections of mid-carotid 4 days afterdenudation. The thoracic aorta is denuded of endothelium using a 2FFogarty arterial embolectomy catheter. The catheter is inserted into thethoracic aorta via the left iliac artery, inflated with 0.2 ml air, andpassed through the lumen five times to remove the endothelium. The iliacartery is then ligated. Three times (3, 7 and 14 days) are selected forevaluation of the histological changes.

To quantify the proliferating cells, 3 different procedures are used forlabelling the cells with bromodeoxyuridine (BrdU) after denudation ofthe rat carotid. In this model, the media cell proliferation begins 24 hafter denudation; cells in the intima first appear after 72-96 hours. Toquantify the proliferation of smooth-muscle cells before the appearanceof cells in the intima, 0.1 ml BrdU-labelling reagent (ZYMED, SanFrancisco, Calif.) is administered i.v. during the postoperative periodof 0 to 72 h post-denudation (in total 0.1 ml 6 times). To quantify theproliferation during the initial wave of migration, the rats were given3×0.1 ml BrdU-labelling reagent at 8-hour intervals over a period of72-96 hours after the operation. To quantify the proliferation at theend of the initial wave of migration, a third group of rats is given apulsed dose of 0.3 ml BrdU three hours before sacrifice.

Histological samples are fixed in 3% paraformaldehyde solution for 4 hfor embedding in paraffin. Morphological changes are evaluated fromparaffin sections stained with Mayer's haematoxylin-eosin. The cellcounts of different vessel sections are calculated at a magnification of400×. To identify cells in culture and cells appearing in the neo-intimawithin four days of the denudation injury, immunohistochemical stainingof acetone-fixed samples is carried out using an anti-a-actin antibodyobtained from smooth-muscle cells (Bio-Makor, Rehovot, Israel). Primarysmooth-muscle cells are identified on acetone-fixed glass cover slidesusing the same staining method. The sections are incubated with theprimary antibody (dilution 1:2000), washed, and incubated consecutivelywith peroxidase-conjugated rabbit-antimouse-Ig and goat-antirabbit-Ig,followed by treatment with substrate solution with the chromogen3-amino-9-ethylcarbazol and hydrogen peroxide. BrdU stains are preparedfrom paraffin sections using a primary mouse antibody (Bu20a, Dako, A/S,Denmark) and the Vectastain Elite ABC-Kit (Vector Laboratories,Burliname, Calif.). The sections are deparaffinised and treated bymicrowave at 500 W (2×5 min in 0.1 M citrate buffer, pH 6), followed bytreatment with 95% formamide in 0.15 M trisodium citrate for 45 min at70° C. Antibody dilutions are prepared according to the manufacturer'sspecifications. The sections are counterstained with Mayer'shaematoxylin and eosin, and positive cells are counted separately forthe initima, media, and adventitia.

In the carotid of treated animals, a significant decrease is found inthe cell count for smooth-muscle cells. The adventitia and the mediashowed a significant reduction in the cell count. As a result of acompound of formula I or II, a slight decrease in the absolute number ofBrdU-labelled cells is seen in the intima, media, and adventitia duringthe first two labelling periods (0-72 and 72-96 h), and after 93-96 h adecrease in the number of labelled cells is seen in all compartments.Decreases in the number of smooth-muscle cells are likewise found in theaorta-denuded animals.

According to these findings, a compound of formula I or II can thusinhibit the proliferation, and especially the migration, of vascularsmooth-muscle cells.

A compound of formula I or II is also capable of inhibitingangiogenesis. This may be demonstrated as follows: a chamber containingagar (0.8%) and heparin (2 U/ml) with or without growth factor (VEGF 3μg/ml, PDGF 1 μg/ml or bFGF 0.3 μg/ml) is implanted subcutaneously intonormal mice (C57 BU6). A compound of formula I or II is administeredorally in a dose showing good anti-tumour activity in a nude mousexenotransplant model. Dosing is started one day before implantation ofthe chambers. The chambers are removed after 5 days. The angiogenicefficacy is quantified by measuring both the vascularised tissue whichhas grown around the implant and the blood content of this tissue(external blood). The blood is determined by measuring the haemoglobin.Although the vessels do not grow into the agar, the agar becomesintensely red if an antiangiogenic effect is present. If a compoundinhibits the increase in blood that is induced by the growth factor,this is seen as an indication that the compound in question is blockingthe angiogenic effect of the growth factor concerned. Inhibition of theweight but not the volume of blood suggests an effect on theproliferation of fibroblasts. A suppression of the control responsesuggests an inhibition of wound healing. At an oral dose of 50 mg/kgonce daily, a compound of formula I or II inhibits the angiogenic effectof all three growth factors (VEGF, PDFG, bFGF).

Interestingly, it was found that4-[(4-methyl-1-piperazinyl)-methyl]-N-[4-hydroxymethyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,4-[(4-methyl-4-oxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamideand4-[(4-methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamiderepresent metabolites ofN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide(ST1571 or imatinib, hereinafter compound A) which can be found in thehuman body upon administration of compound A. Compound A Is described inEP 0 564 409 B1 and, in the form of the methane sulfonate salt, in WO99/03854.

In addition to the before-mentioned metabolites, further compound Ametabolites were identified in monkeys such as4-[(4-methylcarbonyl-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-carboxy-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,4-carboxy-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,4-[(4-methyl-1piperazinyl)-methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamidewherein the pyridinyl moiety is substituted at a ring carbon atom byhydroxy, and4-[(1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamidewherein the pyridinyl moiety is substituted at a ring carbon atom byhydroxy.

Preference is given to compounds of formula II, wherein

-   R₁ is hydrogen,-   R₂ is methyl or hydroxymethyl,-   R₃ is methyl, and    the stars indicate the nitrogen atoms which optionally carry an    oxygen atom to form the corresponding N-oxides,    with the proviso that at least one of the three nitrogen atoms    marked by a star carries an oxygen atom if R₂ is methyl,    or salts of such compounds.

Special preference is further given to compounds of formula II, wherein

-   R₁ is hydrogen,-   R₂ is hydroxy-lower alkyl,-   R₃ is methyl, and    the stars indicate the nitrogen atoms which optionally carry an    oxygen atom to form the corresponding N-oxides,    or salts of such compounds.

Especially preferred are the compounds selected from4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-hydroxymethyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,4-[(4-methyl-4-oxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamideand4-[(4-methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide,and pharmaceutically acceptable salts of these compounds.

Very special preference is further given to a compound of formula I orII mentioned In the Examples below, or a salt, especially apharmaceutically acceptable salt, thereof.

The compounds of formula I or II or salts thereof are prepared inaccordance with processes known per se, though not previously describedfor the manufacture of the compounds of the formula I or II, especiallywhereby

-   a) a compound of formula III    wherein R₁ and R₂ have the meanings given under formula I, is    reacted with a compound of formula IV    wherein A has the meanings given under formula I.    and a compound thus obtained is converted into a N-oxide of formula    I with a suitable oxidizing agent or if not converted into a    N-oxide, A has to be substituted by oxo on a ring carbon;    preferably,    a compound of formula III    wherein R₁ and R₂ have the meanings given under formula II and the    star indicates a nitrogen atom which optionally carries an oxygen    atom, is reacted with a compound of formula IV    wherein R₃ has the meanings given under formula II and the stars    indicate the nitrogen atoms which optionally carry an oxygen atom;    and a compound thus obtained is optionally converted into a N-oxide    of formula II with a suitable oxidizing agent; or-   b) a compound of formula V    wherein R₁ and R₂ have the meanings given under formula I, Hal is    halo (e.g. —Cl, —Br, —F, —I), is reacted with a compound of formula    VI    AH  (VI),    wherein A has the meanings given under formula I,    and a compound thus obtained is optionally converted into a N-oxide    of formula I with a suitable oxidizing agent;    preferably, a compound of formula V    wherein R₁ and R₂ have the meanings given under formula II, Hal is    halo (e.g. —Cl, —Br, —F, —I) and the star indicates a nitrogen atom    which optionally carries an oxygen atom, is reacted with a compound    of formula VI    wherein R₃ has the meanings given under formula II and the stars    indicate the nitrogen atoms which optionally carry an oxygen atom;    and a compound thus obtained is optionally converted into a N-oxide    of formula II with a suitable oxidizing agent;    whereby functional groups which are present in the starting    compounds of process a) or b) and are not intended to take part in    the reaction, are present in protected form if necessary, and    protecting groups that are present are cleaved, whereby the said    starting compounds may also exist in the form of salts provided that    a salt-forming group is present and a reaction in salt form is    possible;    and, if so desired, a compound of formula I or II obtained by    process a) or b) is converted into another compound of formula I or    II, an obtained free compound of formula I or II is converted into a    salt, an obtained salt of a compound of formula I or II is converted    into the free compound or another salt, and/or a mixture of isomeric    compounds of formula I or II is separated into the individual    isomers.

In a most preferred embodiment, compounds of formula I or II are in asubstantially pure form.

The term “substantially pure” is understood in the context of thepresent invention to mean substantially free of biological material suchas found in the blood, especially less than 10%, preferably less than1%, and most preferably free of such biological material.

Description of the Process Variants

A suitable oxidizing agent for converting a compound obtained by processa) or b) into a N-oxide of formula I or II is preferably hydrogenperoxide or a suitable peracid, for example a suitable perbenzoic acid,such as especially m-chloro-perbenzoic acid. The reaction is carried outin an inert solvent, for example a halogenated hydrocarbon, such asdichloromethane, at temperatures of approximately from −20° C. to theboiling point of the solvent in question, in general below +100° C. Ifhydrogen peroxide is used as the oxidizing agent, the reaction ispreferably carried out in water at about room temperature. The desiredN-oxide can then be purified using conventional methods such as e.g.column chromatography or recrystallisation.

On the other hand, the N-oxides of formula I or II may be preparedaccording to the process described in the preceding paragraph by alreadyoxidizing the starting materials used in the synthesis of compounds offormula I or II.

Regarding Process a):

The reaction between a compound of formula III and a compound of formulaIV preferably takes place in a suitable inert solvent, especiallyN,N-dimethylformamide, in the presence of propylphosphonic anhydride(Fluka, Buchs, Switzerland) and a base such as especially triethylamine,preferably at room temperature.

Regarding Process b):

The reaction between a compound of formula V and a compound of formulaIV preferably takes place in a suitable inert solvent, especiallyalcohols, e.g. lower alcohols such as especially ethanol, at elevatedtemperature, preferably near the boiling temperature of the solventemployed.

Halo present in a compound of formula V is e.g. fluoro, chloro, bromoand iodo, preferably chloro.

Additional Process Steps

In the additional process steps, carried out as desired, functionalgroups of the starting compounds which should not take part in thereaction may be present in unprotected form or may be protected forexample by one or more protecting groups. The protecting groups are thenwholly or partly removed according to one of the known methods.

Protecting groups, and the manner in which they are introduced andremoved are described, for example, in “Protective Groups in OrganicChemistry”, Plenum Press, London, New York 1973, and in “Methoden derorganischen Chemie”, Houben-Weyl, 4th edition, Vol. 15/1,Georg-Thieme-Verlag, Stuttgart 1974 and in Theodora W. Greene,“Protective Groups in Organic Synthesis”, John Wiley & Sons, New York1981. A characteristic of protecting groups is that they can be removedreadily, i.e. without the occurrence of undesired secondary reactions,for example by solvolysis, reduction, photolysis or alternatively underphysiological conditions.

The end products of formula I or II may however also containsubstituents that can also be used as protecting groups in startingmaterials for the preparation of other end products of formula I or II.Thus, within the scope of this text, only a readily removable group thatis not a constituent of the particular desired end product of formula Iis designated a “protecting group”, unless the context indicatesotherwise.

General Process Conditions

All process steps described here can be carried out under known reactionconditions, preferably under those specifically mentioned, in theabsence of or usually in the presence of solvents or diluents,preferably those that are inert to the reagents used and able todissolve them, in the absence or presence of catalysts, condensingagents or neutralising agents, for example ion exchangers, typicallycation exchangers, for example in the protonated (H⁺-) form, dependingon the type of reaction and/or reactants at reduced, normal, or elevatedtemperature, for example in the range from −100° C. to about 190° C.,preferably from about −80° C. to about 150° C., for example at −80 to−60° C., at RT, at −20 to 40° C., at 0 to 100° C. or at the boilingpoint of the solvent used, under atmospheric pressure or in a closedvessel, if need be under pressure, and/or in an inert, for example anargon or nitrogen, atmosphere.

The invention relates also to those embodiments of the process in whichone starts from a compound obtainable at any stage as an intermediateand carries out the missing steps, or breaks off the process at anystage, or forms a starting material under the reaction conditions, oruses said starting material in the form of a reactive derivative orsalt, or produces a compound obtainable by means of the processaccording to the invention under those process conditions, and furtherprocesses the said compound in situ. In the preferred embodiment, onestarts from those starting materials which lead to the compoundsdescribed hereinabove as preferred.

In the preferred embodiment, a compound of formula I or II is preparedaccording to the processes and process steps defined in the Examples.

The compounds of formula I or II, including their salts, are alsoobtainable in the form of hydrates, or their crystals can include forexample the solvent used for crystallisation (present as solvates).

Starting Materials

New starting materials and/or intermediates, as well as processes forthe preparation thereof, are likewise the subject of this invention. Inthe preferred embodiment, such starting materials are used and reactionconditions so selected as to enable the preferred compounds to beobtained.

The starting materials used in the above described process are known,capable of being prepared according to known processes (see also EP 0564 409 B1), or commercially obtainable; in particular, they can beprepared using processes as described in the Examples.

In the preparation of starting materials, existing functional groupswhich do not participate in the reaction should, if necessary, beprotected. Preferred protecting groups, their introduction and theirremoval are described above or in the Examples. In place of therespective starting materials and transients, salts thereof may also beused for the reaction, provided that salt-forming groups are present andthe reaction with a salt is also possible. Where the term startingmaterials is used hereinbefore and hereinafter, the salts thereof arealways included, insofar as reasonable and possible.

A compound of formula III wherein R₂ is lower alkyl and the nitrogenatom marked by a star does not carry an oxygen atom as a substituent canbe prepared as described in EP 0 564 409 B1. Such compounds may then beconverted into the corresponding N-oxides using a suitable oxidizingagent as described above under “Description of the process variants”.

A compound of formula III wherein R₂ is hydroxy-lower alkyl can beprepared analogously to Example 1 by starting with a compound of thefollowing formula VII:

The remaining starting materials are known, capable of being preparedaccording to known processes like those described in e.g. EP 0 564 409B1, or commercially available; or in particular, they can be preparedusing processes as described in the Examples. SuitableN-phenyl-2-pyrimidine-amine derivatives to form the correspondingN-oxides are also described in e.g. EP 0 564 409 B1.

The invention relates also to a process for the treatment ofwarm-blooded animals, including humans, suffering from said diseases,especially a tumour disease, wherein a quantity of a compound of formulaI or II which is effective against the disease concerned, especially aquantity with antiproliferative and especially tumour-inhibitingefficacy, is administered to warm-blooded animals, Including humans, inneed of such treatment. The invention relates moreover to the use of acompound of formula I or II for the inhibition of the above-mentionedtyrosine kinases, especially PDGF receptor kinase, v-Abl kinase, and/orc-Kit receptor kinase, or for the preparation of pharmaceuticalcompositions for use in treating warm-blooded animals, including humans,especially for the treatment of tumours, such as gliomas, ovariantumours, prostate tumours, colon tumours, and tumours of the lung, suchas especially small cell lung carcinoma, and tumours of the breast orother gynaecological tumours. Depending on species, age, individualcondition, mode of administration, and the clinical picture in question,effective doses, for example daily doses of about 1-2500 mg, preferably1-1000 mg, especially 5-500 mg, are administered to warm-bloodedanimals, including humans, of about 70 kg bodyweight.

Thus, in a further aspect, the present invention relates to the use ofN-phenyl-2-pyrimidine—amine derivatives in which at least one nitrogenatom carries an oxygen atom to form the corresponding N-oxides or apharmaceutically acceptable salt of such a compound for the preparationof a pharmaceutical composition for the treatment of a proliferativedisorder.

Preferably, the present invention relates to the use of a compound offormula I or II or a pharmaceutically acceptable salt of such a compoundfor the preparation of a pharmaceutical composition for the treatment ofa proliferative disorder.

Most preferably, the proliferative disorder is selected from tumors orbrain proliferative disorders.

The invention further provides a method of treating warm-bloodedanimals, including humans, which comprises administering to such awarm-blooded animal suffering from a proliferative disorder, in a doseeffective against said disorder, a compound of formula I or II or apharmaceutically acceptable salt of such a compound.

In still another embodiment, the instant invention provides apharmaceutical composition comprising at least oneN-phenyl-2-pyrimidine-amine derivative in which at least one nitrogenatom carries an oxygen atom to form the corresponding N-oxides or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier.

Preferably, the instant invention provides a pharmaceutical compositioncomprising a compound of formula I or II or a pharmaceuticallyacceptable salt thereof, together with a pharmaceutically acceptablecarrier.

The compositions of the present invention may contain at least oneadditional pharmaceutically active compound such as4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamide.

Preferably a pharmaceutical composition for the treatment of aproliferative disorder in warm-blooded animals, including humans,comprising as an active ingredient a compound of formula I or IIaccording or a pharmaceutically acceptable salt of such a compound,together with a pharmaceutically acceptable carrier.

Thus the invention relates also to pharmaceutical compositionscomprising as an active ingredient a compound of formula I or IItogether with a pharmaceutically acceptable carrier, especially for theprevention or treatment of one of the said diseases, said pharmaceuticalcompositions being suitable for e.g. topical, enteral, for example oralor rectal, or parenteral administration. Especially tablets or gelatincapsules containing the active substance together with diluents, forexample lactose, dextrose, sucrose, mannitol, sorbitol, cellulose,and/or glycerin, and/or lubricants, for example silica, talc, stearicacid, or salts thereof, typically magnesium or calcium stearate, and/orpolyethylene glycol, are used for oral administration. Tablets maylikewise contain binders, for example magnesium aluminium silicate,starches, typically corn, wheat or rice starch, gelatin,methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, and, if so desired, disintegrants, for examplestarches, agar, alginic acid, or a salt thereof, typically sodiumalginate, and/or effervescent mixtures, or adsorbents, colouring agents,flavours, and sweetening agents. The pharmacologically active compoundsof the present invention may further be used in the form of preparationsfor parenteral administration or infusion solutions. Such solutions arepreferably isotonic aqueous solutions or suspensions, these possiblybeing prepared before use, for example in the case of lyophilisedpreparations containing the active substance either alone or togetherwith a carrier, for example mannitol. The pharmaceutical substances maybe sterilised and/or may contain excipients, for example preservatives,stabilisers, wetting agents and/or emulsifiers, solubilisers, salts forthe regulation of osmotic pressure, and/or buffers. The presentpharmaceutical compositions which, if so desired, may contain furtherpharmacologically active substances, such as other c-Kit inhibitors orinhibitors of VEGF receptor or c-Src activity, are prepared in a mannerknown per se, for example by means of conventional mixing, granulating,coating, dissolving or lyophilising processes, and contain from about 1%to 100%, especially from about 1% to about 20%, of the active substanceor substances.

EXAMPLES

The following Examples illustrate the invention but do not limit thescope thereof in any way.

Abbreviations:

-   DMF N,N-Dimethylformamide-   h hour(s)-   min minute(s)-   m.p. melting point-   RT room temperature-   THF tetrahydrofuran

Example 14-[(4-Methyl-1-piperazinvi)-methyl]-N-[4-hydroxymethyl-3-{[4-(3-pyridinyl)-2-Pnrimidinyl]-amino]-phenyl]-benzamide

A solution of propylphosphonic anhydride in N,N-dimethylformamide(Fluka, Buchs, Switzerland; 350 μL of 50%, 0.6 mmol) is added inportions over 20 min to a stirred mixture ofN-(5-amino-2-hydroxymethyl-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine (117mg, 0.4 mmol), 4-[(4-methyl-1-piperazinyl)-methyl]-benzoic aciddihydrochloride (123 mg, 0.4 mmol) and triethylamine (445 μL, 3.2 mmol)in dry N,N-dimethylformamide (5 mL). The mixture is stirred for 24 h atRT. The solvent is evaporated off under reduced pressure and the residueis treated with saturated aqueous sodium hydrogen carbonate solution (20mL) and extracted with ethyl acetate (2×20 mL). The combined extractsare washed with saturated aqueous sodium chloride (15 mL), dried(MgSO₄), filtered and the solvent is evaporated off under reducedpressure to yield the crude product which is purified by reverse phasehigh pressure liquid chromatography (Nagel Polygoprep C₁₈, 7 μm, 300 Å;Macherey-Nagel, Duren, Germany), eluent 0.1% trifluoroacetic acid inwater −0.1% trifluoroacetic acid in acetonitrile. The fractionscontaining the pure product are combined, basified with saturatedaqueous sodium hydrogen carbonate and evaporated to dryness underreduced pressure. The residue is treated with saturated aqueous sodiumhydrogen carbonate and extracted with ethyl acetate (5×). The combinedextracts are washed with water, dried (MgSO₄), filtered and the solventis evaporated off under reduced pressure to yield the product which isrecrystallised from methanol—ethyl acetate to give the title compound asa pale-yellow crystalline solid, m.p. 196-198° C.

¹H-NMR (500 MHz, DMSO-d₆, δ): 2.14 (s, 3H), 2.25-2.45 (m, 8H), 3.52 (s,2H), 4.56 (s, 2H), 5.50 (br.s, 1H), 7.29 (d, J=8.3 Hz, 1H), 7.41 (dd,J=2.0, 8.3 Hz, 1H), 7.44 (d, J=8.1 Hz, 2H), 7.50 (d, J=5.1 Hz, 1H), 7.52(dd, J=3.3, 8.1 Hz, 1H), 7.93 (d, J=8.1 Hz, 2H), 8.56 (d, J=2.0 Hz,1H),8.57 (d, J=5.1 Hz,1H), 8.59 (ddd, J=1.4, 2.1, 8.1 Hz,1H), 8.69 (dd,J=1.4, 3.3 Hz, 1H), 9.10 (s, 1H), 9.33 (d, J=2.1 Hz, 1H) and 10.22 (s,1H).

Step 1.1: 2-Amino-4-nitrobenzenemethanol

A stirred solution of 2-amino-4-nitrobenzoic acid (Aldrich; 18.2 g, 100mmol) in dry THF (500 mL) at 200C, is treated with a solution ofborane-THF complex (BH₃-THF; Fluka; 100 mL of 1.0 M), dropwise over 45min to regulate the gas evolution. The mixture is then heated at 650Cfor 2 h. The stirred mixture is then cooled to 0° C., treated with water(20 mL) and warmed to RT. Upon the cessation of gas evolution,hydrochloric acid (20 mL of 12 M) is added and the mixture is thenheated at 650C for 30 min. The cooled mixture is then concentrated to avolume of circa 150 mL by rotary evaporation under reduced pressure togive a suspension. The suspension is filtered and the precipitate isredissolved in ethyl acetate (500 mL) and washed with saturated aqueoussodium hydrogen carbonate (2×150 mL). The solution is dried (Na₂SO₄),filtered and the solvent is evaporated off under reduced pressure toyield the crude product which is purified by recrystallisation fromethyl acetate-hexane to give the title compound as a yellow crystallinesolid, m.p. 126-128° C.

Step 1.2: 2-[(2-Propenyloxy)-methyl]-5-nitrobenzenamine

A stirred solution of 2-amino-4-nitrobenzenemethanol (14.3 g, 85 mmol)in dry THF (350 mL) at 0° C. under an argon atmosphere, is treateddropwise over 35 min with a solution of potassium tert-butylate in THF(Fluka; 85 mL of 1.0 M). The mixture is stirred at 0° C. for 15 min andthen treated dropwise over 50 min with a solution of allylbromide (7.9mL, 94 mmol) in dry THF (80 mL) at 0° C. and then stirred at 200C for 90min. The mixture is diluted with ethyl acetate (800 mL). The resultingsolution is washed with saturated aqueous ammonium chloride (3×400 mL),dried (MgSO₄), filtered and the solvent Is evaporated off under reducedpressure to yield the crude product which is purified by columnchromatography on silica gel, eluent 50% ethyl acetate in hexane, togive the title compound as a brown oil.

¹H-NMR (500 MHz, DMSO-d₈, 8): 4.06 (d, J=5.3 Hz, 2H); 4.47 (s, 2H); 5.22and 5.34 (dd, J=10.4, 17.3 Hz, 2H); 5.65 (br.s, 2H); 5.98 (m, J=5.3,10.4, 17.3 Hz,1H), 7.35 (d, J=8.3 Hz, 1H), 7.38 (dd, J=2.0, 8.3 Hz, 1H)and 7.52 (d, J=2.0 Hz, 1H).

Step 1.3: {2-[(2-Propenyloxy)-methyl]-5-nitrophenyl}-guanidine

Nitric acid (1.04 mL of 65%, 15 mmol) is added to a stirred solution of2-[(2-propenyloxy)-methyl]-5-nitrobenzenamine (3.15 g, 15 mmol) inethanol (30 mL) at 200C. A solution of cyanamide (0.95 g, 22.5 mmol) inwater (1 mL) is then added dropwise to the stirred mixture at 95° C.over a period of 60 min. The mixture is heated at 950C for 14 h, withadditional aliquots of cyanamide (total 2.2 g, 58 mmol) being addedthroughout this period and with the acidity being periodically adjustedto pH 3 by the addition of nitric acid (65%). The resulting mixture iscooled to 0° C., basified with aqueous ammonia (5 mL of 25%), dilutedwith water (150 mL) and extracted with ethyl acetate (3×100 mL). Thecombined extracts are washed with saturated aqueous ammonium chloride(50 mL), dried (MgSO₄), filtered and the solvent is evaporated off underreduced pressure to give the title compound as a brown oil, which isused directly in the next step without further purification.

Step 1.4:N-[2-[(2-Propenyloxy)-methyl]-5-nitro-phenyl}-4-(3-pyridinyl)-2-pyrimidinamine

A stirred mixture of{2-[(2-propenyloxy)-methyl]-5-nitro-phenyl}-guanidine (3.75 g, 15 mmol),3-(dimethylamino)-1-(3-pyridinyl)-2-propen-1-one (2.60 g, 15 mmol) andethyl diisopropylamine (2.6 mL, 15 mmol) in 1-butanol (50 mL) is heatedat 1200C for 20 h. The solvent is then evaporated off under reducedpressure to give a residue which is dissolved in ethyl acetate (100 mL).The resulting mixture is filtered (celite), washed with saturatedaqueous sodium chloride (50 mL), dried (MgSO₄), filtered and the solventis evaporated off under reduced pressure to yield the crude productwhich is purified by column chromatography on silica gel, eluent ethylacetate, and recrystallised from ethyl acetate-hexane to give the titlecompound as a yellow crystalline solid, m.p. 213-2150C.

¹H-NMR (500 MHz, DMSO-d₆, δ): 4.10 (d, J=5.3 Hz, 2H); 4.77 (s, 2H); 5.22and 5.35 (dd, J=10.4, 17.3 Hz, 2H); 5.96 (m, J=5.3,10.4, 17.3 Hz, 1H),7.58 (dd, J=4.8, 7.9 Hz, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.69 (d, J=5.2 Hz,1H), 7.95 (ddd, J=1.2, 1.2, 7.9 Hz, 1H), 8.51 (dd, J=1.6, 8.4 Hz, 1H),8.67 (d, J=5.2 Hz, 1H), 8.73 (dd, J=1.2, 4.8 Hz, 1H), 9.05 (d, J=1.2 Hz,1H), 9.23 (br.s, 1H) and 9.35 (d, J=1.6 Hz, 1H).

Step 1.5:N-(2-Hydroxymethyl-5-nitro-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine

Polymethylhydrosiloxane (860 mg), tetrakis(triphenylphoshine)palladium(70 mg) and zinc chloride (2.66 mL of 0.5 M in THF, 1.33 mmol) is addedto a stirred solution ofN-{2-[(2-propenyloxy)-methyl]-5-nitro-phenyl}-4-(3-pyridinyl)-2-pyrmidinamine(2.60 g, 7.2 mmol) in dry THF (60 mL). The mixture is then stirred underan argon atmosphere at 30° C. for 30 h. The solvent is then evaporatedoff under reduced pressure to give a residue which is treated withsaturated aqueous sodium chloride solution (50 mL) and extracted withethyl acetate (3×50 mL). The combined extracts are dried (Na₂SO₄),filtered and the solvent is evaporated off under reduced pressure toyield the crude product which is recrystallised from THF to give thetitle compound as a pale-yellow crystalline solid, m.p. 247-250° C.

Step 1.6:N-(5-Amino-2-hydroxymethyl-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine

A solution ofN-(2-hydroxymethyl-5-nitro-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine(0.23 g, 0.71 mmol) in ethanol (230 mL) is hydrogenated at atmosphericpressure over Raney nickel (0.2 g) at 250C. The calculated amount ofhydrogen is taken up in 13 h. The mixture is then filtered and thesolvent is evaporated off under reduced pressure to yield the crudeproduct which is purified by column chromatography on silica gel, eluent25% aqueous ammonia-ethanol-dichloromethane (1:9:90), to give the titlecompound as a yellow crystalline solid, m.p. 213-215° C.

¹H-NMR (500 MHz, DMSO-d₆, δ): 4.42 (d, J=5.1 Hz, 2H), 5.05 (br.s, 2H),5.26 (t, J=5.1 Hz, 1H), 6.23 (dd, J=2.1, 8.0 Hz, 1H), 6.91 (d, J=8.0 Hz,1H), 7.35 (d, J=2.0 Hz, 1H), 7.45 (d, J=5.1 Hz, 1H), 7.56 (dd, J=4.7,8.0 Hz, 1H), 8.47 (ddd, J=1.8, 1.8, 8.0 Hz, 1H), 8.53 (d, J=5.1 Hz, 1H),8.70 (dd, J=1.4, 4.7 Hz, 1H), 8.88 (s, 1H) and 9.29 (d, J=2.4 Hz, 1H).

Example 24-[(4-Methyl-4-oxido-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2Pyrimidinyl]-amino]-phenyl]-benzamide

3-Chloroperoxybenzoic acid (Fluka, Buchs, Switzerland; 2.06 g of 55%,4.27 mmol) is added to a stirred mixture of4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl)-benzamide(prepared as described in EP 0 564 409 B1, Example 21; 2.00 g, 4.05mmol) in dichloromethane (70 mL) at −20° C. The resulting mixture isthen stirred at RT for 72 h. The solvent is then evaporated off underreduced pressure to yield a mixture which is purified by columnchromatography on silica gel, eluent dichloromethane-methanol-water(70:30:5), to give the title compound as a yellow crystalline solid,m.p. 154-158° C.

Example 34-[(4-Methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-{[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide

N-Methylpiperazine (99 mg, 1.0 mmol) is added to a stirred suspension of4-chloromethyl-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide(220 mg, 0.49 mmol) In ethanol (5 mL). The mixture is then stirred at100C for 15 h to give a solution, which is then cooled to RT and treatedwith ethyl acetate (200 mL). The resulting solution is washed withaqueous sodium hydroxide (100 mL of 2M) and saturated aqueous sodiumchloride solution (100 mL), dried (Na₂SO₄), filtered and the solvent isevaporated off under reduced pressure to yield the crude product whichis purified by column chromatography on silica gel, eluent 25% aqueousammonia-methanol-dichloromethane (0.5:10:90) to give the title compoundas a yellow crystalline solid, m.p. 232-235° C.

Step 3.1:N-[4-Methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide

Utilising the procedure described in Example 2, but employingN-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide(prepared as described in EP 0 564 409 B1, Example 20) in place of4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,afforded the title compound which is purified by column chromatographyon silica gel, eluent 10% methanol in dichloromethane, andrecrystallised from ethanol to give the title compound as a pale-yellowcrystalline solid, m.p. 258-260° C.

Step 3.2:4-Methyl-N-3-[4-(1-oxido-3-pyridinyl]-2-pyrimidinyl-1.3-benzenediamine

Hydrochloric acid (9 mL of 4M) is added to a suspension ofN[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide(0.43 g, 1.08 mmol) in n-propanol (9 mL) and the resulting mixture isheated at 100° C. for 34 h. The cooled mixture is evaporated underreduced pressure to give an oil, which is dissolved in water (10 mL),filtered and basified with aqueous sodium hydroxide (4 M). The resultingprecipitate is filtered, washed with water and dried to yield the crudeproduct, which is recrystallised from ethanol to give the title compoundas a yellow crystalline solid, m.p. 104-106° C.

Step 3.3:4-Chloromethyl-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide

A solution 4-(chloromethyl)-benzoyl chloride (Fluka, Buchs, Switzerland;184 mg, 0.977 mmol) in dioxane (2 mL) is added dropwise to a solution of4-methyl-N-3-[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-1,3-benzenediamine(275 mg, 0.937 mmol) in dioxane (5 mL) and the mixture is stirred at 20°C. for 75 min. A second portion of 4-(chloromethyl)-benzoyl chloride (60mg, 0.317 mmol) dissolved in dioxane (1 mL) is then added and themixture is stirred for a further 120 min. The resulting suspension istreated with ethyl acetate (50 mL) to give a solution which is washedwith aqueous sodium hydroxide (2×50 mL of 2M). The ethyl acetatesolution is dried (Na₂SO₄), filtered and the solvent is evaporated offunder reduced pressure to yield the crude product which is purified bycolumn chromatography on silica gel, eluent 5% methanol indichloromethane to give the title compound as a yellow crystallinesolid, m.p. 224-226° C.

Example 44-[(4-Methyl-1.4-dioxido-1-piperazinyl)-methyl]-N-4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide

4-[(4-Methyl-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamidemonomethanesulphonate (3.00 g, 5 mmol; prepared as described in WO99/03854) is added to aqueous hydrogen peroxide (30 mL of 3%) and theresulting solution is stirred at 20° C. for 160 h. The pH of thesolution is then adjusted to pH 14 with aqueous sodium hydroxide (4 M)and the resulting suspension is stirred for 1.5 h. The crude product isfiltered off, washed with water, dried and purified by columnchromatography on silica gel, eluent 25% aqueousammonia-ethanol-dichloromethane (5:30:70), to give the title compound asa yellow crystalline solid, m.p. 242-244° C.

Example 54-[(4-methyl-1-piperazinyl)methyl]-N-[4-hydroxymethyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide

A solution of propylphosphonic anhydride in N,N-dimethylformamide(Fluka, Buchs, Switzerland; 350 μL of 50%, 0.6 mmol) is added inportions over 20 minutes to a stirred mixture ofN-[2-[5-amino-(2-hydroxy)methyl]phenyl}-4-(3-pyridinyl)-2-pyrimidinamineas described in example 1, step 1.6 (117 mg, 0.4 mmol),4-[(4-methyl-1-piperazinyl)methyl]benzoic acid, dihydrochloride (123 mg,0.4 mmol) and triethylamine (445 μL, 3.2 mmol) in dryN,N-dimethylformamide (5 mL). The mixture is stirred for 24 hours atroom temperature. The solvent is evaporated off under reduced pressureand the residue is treated with saturated aqueous sodium hydrogencarbonate solution (20 mL) and extracted with ethyl acetate (2×20 mL).The combined extracts are washed with saturated aqueous sodium chloride(15 mL), dried (MgSO₄), filtered and the solvent is evaporated off underreduced pressure to yield the crude product which is purified by reversephase high pressure liquid chromatography (Nagel Polygoprep C₁₈, 7 μm,300 Å; Macherey-Nagel, DOren, Germany), eluent 0.1% trifluoroacetic acidin water −0.1% trifluoroacetic acid in acetonitrile. The fractionscontaining the pure product are combined, basified with saturatedaqueous sodium hydrogen carbonate and evaporated to dryness underreduced pressure. The residue is treated with saturated aqueous sodiumhydrogen carbonate and extracted with ethyl acetate (5×). The combinedextracts are washed with water, dried (MgSO₄), filtered and the solventis evaporated off under reduced pressure to yield the product which isrecrystallised from methanol—ethyl acetate to give the title compound asa pale-yellow crystalline solid, m.p. 196-198° C.

¹H-NMR (500 MHz, DMSO-d₆, δ):2.14 (s, 3H), 2.25-2.45 (m, 8H), 3.52 (s,2H), 4.56 (s, 2H), 5.50 (br.s, 1H), 7.29 (d, J=8.3 Hz, 1H), 7.41 (dd,J=2.0, 8.3 Hz, 1H), 7.44 (d, J=8.1 Hz, 2H), 7.50 (d, J=5.1 Hz, 1H), 7.52(dd, J=3.3, 8.1 Hz, 1H), 7.93 (d, J=8.1 Hz, 2H), 8.56 (d, J=2.0 Hz, 1H),8.57 (d, J=5.1 Hz, 1H), 8.59 (ddd, J=1.4, 2.1, 8.1 Hz, 1H), 8.69 (dd,J=1.4, 3.3 Hz, 1H), 9.10 (s, 1H), 9.33 (d, J=2.1 Hz,1H) and 10.22(s,1H).

Example 64-[(3-Oxo-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide

A solution of propylphosphonic anhydride in N,N-dimethylformamide(Fluka, Buchs, Switzerland; 1.3 mL of 50%, 2.25 mmol) is added dropwiseto a stirred mixture of4-methyl-N-[4-(3-pyridinyl)-2-pyrimidinyl]-1,3-benzenediamine (416 mg,1.5 mmol), 4-[(3-oxo-1-piperazinyl)methyl]benzoic acid (351 mg, 1.5mmol) and triethylamine (1.7 mL, 12 mmol) in dry N,N-dimethylformamide(4 mL). The mixture is stirred for 17 hours at room temperature and thentreated with saturated aqueous sodium hydrogen carbonate solution (100mL). The resulting precipitate is filtered, washed with water, dried andrecrystallised from methanol to give the title compound as a creamcrystalline solid.

¹H-NMR (500 MHz, DMSO-d₆, δ) ppm 2.21 (s, 3H), 2.63 (t, J=5.49 Hz, 2H),2.80 (s, 3H), 2.96 (s, 2H), 3.26 (t, J=5.42 Hz, 2H), 3.60 (s, 2H), 7.19(d, J=8.39 Hz, 1H), 7.42 (d, J=5.19 Hz,1H), 7.46 (m, 3H), 7.51 (dd,J=7.93, 4.73 Hz, 1H), 7.91 (d, J=8.09,1H), 8.06 (s, 1H), 8.47 (m, 1H),8.50 (d, J=5.04 Hz, 1H), 8.67 (dd, J=4.73, 1.53 Hz,1H), 8.99 (s, 1H),9.26 (d, J=1.98 Hz, 1H) and 10.18 (s, 1H).

4-[(3-Oxo-1-piperazinyl)methyl]benzoic acid

A mixture of 3-bromomethylbenzoic acid (4.30 g, 20 mmol),piperazin-2-one (2.0 g, 20 mmol) and powdered potassium carbonate (2.76g, 20 mmol) in methanol (50 mL) is stirred for 17 hours at roomtemperature. The resulting mixture is filtered and the solvent isevaporated off under reduced pressure to give a residue which is treatedwith hydrochloric acid (80 mL of 0.25 M) and stirred for 5 min. Theprecipitated product is filtered, washed with water, dried andrecrystallised from methanol to give the title compound as a creamcrystalline solid.

Example 74-[(4-Methyl-3-oxo-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-Pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide

Utilising the procedure described for Example 6, but employing4-[(4-methyl-3-oxo-1-piperazinyl)methyl]benzoic acid in place of4-[(3-oxo-1-piperazinyl)methyl]benzoic acid afforded the title compoundas a yellow crystalline solid, m.p. 187-192° C.

¹H-NMR (500 MHz, DMSO-d₆, δ) 2.21 (s, 3H), 2.55 (t, J=5.34 Hz, 2H), 2.91(s, 2H), 3.15 (m, 2H), 3.61 (s, 2H), 7.19 (d, J=8.24 Hz, 1H), 7.42 (d,J=5.19 Hz, 1H), 7.46 (t, J=8.01 Hz, 1H), 7.48 (m, 2H), 7.51 (dd, J=7.86,4.81 Hz, 1H), 7.77 (s, 1H), 7.91 (d, J=8.09 Hz, 2H), 8.07 (s, 1H), 8.47(d, J=7.94 Hz, 1H), 8.50 (d, J=5.19 Hz, 1H), 8.67 (d, J=3.36 Hz, 1H),8.99 (s, 1H), 9.27 (s, 1H) and 10.18 (s, 1H).

4-[(4-Methyl-3-oxo-1-piperazinyl)methyl]benzoic acid

Utilising the procedure described for4-[(3-oxo-1-piperazinyl)methyl]benzoic acid, but employing1-methylpiperazin-2-one in place of piperazin-2-one afforded the titlecompound as cream crystalline solid.

Example 8

Tablets containing 100 mg of a compound of formula II, for example oneof the compounds of formula II described in the Examples 1-4, areusually prepared in the following composition:

Composition: Active ingredient 100 mg Crystalline lactose 240 mg Avicel 80 mg PVPPXL  20 mg Aerosil  2 mg Magnesium stearate  5 mg 447 mg

Preparation: The active substance is mixed with carrier materials andcompressed on a tableting machine (Korsch EKO, punch diameter 10 mm).

Avicel is microcrystalline cellulose (FMC, Philadelphia, USA).

PVPPXL is polyvinylpolypyrrolidone, cross-linked (BASF, Germany).

Aerosil is silicon dioxide (Degussa, Germany).

Example 9

Capsules containing 100 mg of a compound of formula II, for example oneof the compounds of formula II described in the Examples 1-4, areusually prepared in the following composition:

Composition: Active ingredient 100 mg Avicel 200 mg PVPPXL 15 mg Aerosil2 mg Magnesium stearate 1.5 mg 318.5 mg

Preparation: The capsules are prepared by mixing the components andfilling the mixture into hard gelatin capsules, size 1.

1-20. (canceled)
 21. (A compound of formula I

wherein R₁ is hydrogen or hydroxy, R₂ is hydrogen, lower alkyl orhydroxy-lower alkyl, A is —NR₅R₆, —CHR₅R₆ or —OR₅R₆, R₅R₆ together isalkylene with four, five or six carbon atoms, oxa-lower alkylene withone oxygen and three or four carbon atoms, or aza-lower alkylene withone or two nitrogen and two, three or four carbon atoms wherein thenitrogen atom is unsubstituted or substituted by lower alkyl,hydroxy-lower alkyl, or acetyl, and wherein lower alkylene in each casemay be partially or totally unsaturated and/or the carbon atoms of loweralkylene may be substituted by lower alkyl, hydroxyl, lower alkoxy oroxo group when lower alkylene is not totally unsaturated, and wherein atleast one nitrogen atom carries an oxygen atom to form the correspondingN-oxide or when no nitrogen atom carries an oxygen atom, A issubstituted by oxo, or a pharmaceutically acceptable salt of such acompound.
 22. A compound of formula I according to claim 21, wherein Ais pyrrolidino, piperidyl, piperidino, piperazinyl, pyridyl,pyrrolidinyl, morpholino, lower alkylpiperazino, N-methylpiperazino,4-methyl-3-oxo-1-piperazinyl, 3-oxo-1-piperazinyl, 1H-imidazolyi,1H-2-methylimidazolyl, 1H-4-methylimidazolyl, 1H-2,4-dimethylimidazolyl,cyclohexyl or phenyl, optionally substituted by oxo on a ring carbon, ora pharmaceutically acceptable salt of such a compound.
 23. A compound offormula I according to claim 21, wherein A is a piperazinyl group of thefollowing formula A′

optionally substituted by oxo on a ring carbon, and R₃ is hydrogen,lower alkyl or acetyl, or a pharmaceutically acceptable salt of such acompound.
 24. A compound of formula I according to claim 23, wherein R₁is hydrogen, R₂ is hydrogen, methyl or hydroxymethyl, R₃ is methyl orhydrogen, or a pharmaceutically acceptable salt of such a compound. 25.A compound of formula I according to claim 21 which is4-[(3-Oxo-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]pheny]benzamide,or a pharmaceutically acceptable salt thereof.
 26. A compound of formulaI according to claim 21,which is4-[(4-Methyl-3-oxo-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide,or a pharmaceutically acceptable salt thereof.
 27. A compound of formulaII

wherein R₁ is hydrogen or hydroxy, R₂ is lower alkyl or hydroxy-loweralkyl, R₃ is hydrogen, methyl or acetyl, and the stars indicate thenitrogen atoms which optionally carry an oxygen atom to form thecorresponding N-oxides, with the proviso that at least one of the threenitrogen atoms marked by a star carries an oxygen atom if R₁ ishydrogen, R₂ is methyl and R₃ is hydrogen or methyl, or a salt thereof.28. A compound of formula II according to claim 27, wherein R₁ ishydrogen, R₂ is methyl or hydroxymethyl, R₃ is methyl, and the starsindicate the nitrogen atoms which optionally carry an oxygen atom toform the corresponding N-oxides, with the proviso that at least one ofthe three nitrogen atoms marked by a star carries an oxygen atom if R₂is methyl, or a salt thereof.
 29. A compound of formula II according toclaim 27, wherein R₁ is hydrogen, R₂ is hydroxy-lower alkyl, R₃ ismethyl, and the stars indicate the nitrogen atoms which optionally carryan oxygen atom to form the corresponding N-oxides, or a salt thereof.30. A compound of formula II according to claim 27 which is4-[(4-methyl-4-oxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide, or a pharmaceutically acceptable saltthereof.
 31. A compound of formula II according to claim 28 which is4-[(4-methyl-1-piperazinyl)-methyl]-N-[4-methyl-3-[[4-(1-oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide, or a pharmaceutically acceptable saltthereof.
 32. A compound of formula II according to claim 28 which is4-[(4-methyl-1,4-dioxido-1-piperazinyl)-methyl]-N-{4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,or a pharmaceutically acceptable salt thereof.
 33. A compound of formulaII according to claim 28 which is4-[(4-methyl-1-piperazinyl)-methyl]-N-{4-hydroxymethyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl}-benzamide,or a pharmaceutically acceptable salt thereof.
 34. A compound accordingto claim 21 in purified form, or a pharmaceutically acceptable saltthereof.
 35. A compound according to claim 21 or a pharmaceuticallyacceptable salt thereof for use in a method for the therapeutictreatment of warm-blooded animals, including humans.
 36. Apharmaceutical composition comprising a compound according to claim 21,or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier.
 37. A pharmaceutical compositionfor the treatment of a proliferative disorder in warm-blooded animals,including humans, comprising as an active ingredient a compoundaccording to claim 21 or a pharmaceutically acceptable salt of such acompound, together with a pharmaceutically acceptable carrier.
 38. Useof a compound according to claim 21 or a pharmaceutically acceptablesalt of such a compound for the preparation of a pharmaceuticalcomposition for the treatment of a proliferative disorder.
 39. Use of acompound according to claim 21 or a pharmaceutically acceptable salt ofsuch a compound for the treatment of a proliferative disorder.
 40. Amethod of treating warm-blooded animals, including humans, whichcomprises administering to such a warm-blooded animal suffering from aproliferative disorder, in a dose effective against said disorder, acompound according to claim 21 or a pharmaceutically acceptable salt ofsuch a compound.
 41. A process for the preparation of a compound offormula II according to claim 27 or a salt thereof, characterized inthat a compound of formula II

wherein R₁ and R₂ have the meanings as defined for a compound of formulaI according to claim 21 and the star indicates a nitrogen atom whichoptionally carries an oxygen atom, is reacted with a compound of formulaIII

wherein R₃ has the meanings as defined for a compound of formula Iaccording to claim 21 and the stars indicate the nitrogen atoms whichoptionally carry an oxygen atom; and a compound thus obtained isoptionally converted into a N-oxide of formula I with a suitableoxidizing agent; whereby functional groups which are present in thecompounds of formula II and III and are not intended to take part in thereaction, are present in protected form if necessary, and protectinggroups that are present are cleaved, whereby the compounds of formula IIand III may also exist in the form of salts provided that a salt-forminggroup is present and a reaction in salt form is possible; and, if sodesired, a compound of formula I thus obtained is converted into anothercompound of formula I, an obtained free compound of formula I isconverted into a salt, an obtained salt of a compound of formula I isconverted into the free compound or another salt, and/or a mixture ofisomeric compounds of formula I is separated into the individualisomers.